Joakim Rödin

Distribution of Napin and Cruciferin in Developing Rape Seed Embryos

The distribution of napin and cruciferin, the two major storage proteins in rape seed, Brassica napus, has been visualized during seed development by antibody staining of paraffin-embedded and sectioned seeds. The results indicate that the synthesis of both proteins during embryogenesis is strictly regulated with respect to time and tissue. Although the synthesis of napin started a few days earlier than that of cruciferin, both proteins displayed similar patterns in their spatial distributions. They were first detected in the axis, then in the outer cotyledon, and finally in the cells of the inner cotyledon. Both proteins are also present in the endosperm, although in lower amounts. In germinating seeds, napin and cruciferin were rapidly degraded. Within 2 days the amounts had decreased dramatically, and after 4 days hardly any cells contained napin or cruciferin. Biochemical analyses of dissected embryos showed that, for napin as well as for cruciferin, similar levels of polypeptides were found in the axis and cotyledons.

Differential expression of myrosinase gene families

In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominantly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axis contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.

Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families

A gene encoding a subunit of the 12S storage globulin, cruciferin, in Brassica napus (oilseed rape) has been isolated and characterized. The gene consists of about 2200 bp including three short intervening sequences. Primer extension analysis showed that the major transcription start site is located 30 bp 5′ of the predicted ATG start codon. This gene belongs to one of three different major families encoding cruciferin subunits. By use of gene-family-specific probes and Southern blotting analysis the number of genes of the three different cruciferin subtypes in B. napus was estimated.

Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions

To define sequences in the cruciferin gene cru1 promoter of importance for expression, tobacco (Nicotina tabacum L.) plants were transformed with constructs in which the cru1 promoter, in front of the intact cru1 structural gene, was truncated at −1216, −974, −736, −515, −306, −46 and −17 bp relative to the cap-site. Cru1 expression in tobacco seeds was studied by Northern analysis, Western analysis and in-situ hybridizations. Comparisons of the Northern analysis of RNA from tobacco seeds harvested at 18 d after pollination with the Western analysis of protein from mature seeds showed that the regions between −974 to −736 and −306 to −46 were important for the expression of cru1 at an early developmental stage, whereas the regions −736 to −515 and −515 to −306 were important for expression throughout embryogenesis. By investigating the mRNA levels in transgenic seeds at different stages of development, indications were obtained that the two latter regions exerted their effects during the later stages. The in-situ hybridization showed that cru1 mRNA was distributed in parenchyma cells throughout the embryo in seeds expressing constructs −974 and −736. Constructs −515 and −306 showed an expression restricted to the axis or axis and parts of the cotyledons. Sequence comparisons of the cru1 promoter with other storage-protein gene promoters, identified several motifs implicated in gene regulation. Gel retardation assays with synthetic oligonucleotides showed that a region present in both cru1 and BnC1 promoters, a CANNTG motif, an SEF3 motif, an abscisic-acid-responsive element and an RY-like motif interacted specifically in vitro with DNA-binding proteins present in nuclear extracts from seeds of Brassica napus L. harvested 40 d after pollination.

Cruciferin gene families are expressed coordinately but with tissue-specific differences during Brassica napus seed development

The major storage protein in seeds of Brassica napus, the 12S globulin cruciferin, is composed of three different groups of subunits; cru1, cru2/3 and cru4. By using gene family-specific probes, we have investigated the accumulation, rate of synthesis and spatial distribution of transcripts corresponding to the different groups of cruciferin subunits in developing seeds. Cruciferin transcripts derived from different gene families accumulate coordinately to comparable amounts during seed development. The corresponding gene families are, however, transcribed at different rates. Investigation of the spatial distribution of transcripts corresponding to each group of cruciferin subunits in the developing seed by in situ hybridization, revealed that mRNAs of all three types accumulate in both axis and cotyledons. Transcripts derived from cru1 and cru4 gene families show a similar cell specificity and accumulate in a similar spatial manner during seed development. In contrast, mRNAs corresponding to the cru2/3 gene family are expressed with a partly different cell specificity and show a slightly different pattern of accumulation in the axis and cotyledons, with a delayed accumulation in epidermal cells. In the cotyledons, the initial accumulation of this type of cruciferin mRNAs is also distinguished from the two other types. The differences in cell specificity are seen in the root cap and in provascular cells, where mRNAs belonging to the cru2/3 family are absent.

The relationship between mature chains and their precursors of cruciferin, the 12S storage protein of Brassica napus

Abstract Cruciferin (12S globulin) is a large, neutral, oligomeric protein synthesized in rapeseed (Brassica napus) during seed development. It is composed of six subunit pairs. Each pair is synthesized as a precursor which in turn gives rise to one heavy α chain ( M r approx. 30 000) and one light β chain ( M r approx. 20 000). Immunoprecipitations of cruciferin synthesized in vitro from rapeseed mRNA showed that at least three major precursor forms exist. Analyses of mature chains on the other hand showed that there are four different α and four different β chains. The majority of α and β chains of each subunit are disulfide-linked. Using peptide mapping and chromatographic separations of subunits, the relationship between mature α and β chains and their precursors could be established.